Search

ObjectiveTo unravel B-cell receptor (BcR) characteristics in muscle tissues and peripheral blood and gain more insight into BcR repertoire changes in peripheral blood in idiopathic inflammatory myopathies (IIMs), and study how this correlates to the clinical response to IVIG.MethodsNineteen treatment-naive patients with newly diagnosed IIM were prospectively treated with IVIG monotherapy. RNA-based BcR repertoire sequencing was performed in muscle biopsies collected before, and in peripheral blood (PB) collected before and nine weeks after IVIG treatment. Results were correlated to patients’ clinical improvement based on the total improvement score (TIS).ResultsPrior to IVIG treatment, BcR clones found in muscle tissue could be retrieved in peripheral blood. Nine weeks after IVIG treatment, new patient-specific dominant BcR clones appeared in peripheral blood while pre-treatment dominant BcR clones disappeared. The cumulative frequency of all dominant BcR clones before treatment was significantly higher in individuals who responded to IVIG compared with those who did not respond to IVIG, and correlated with a higher CK. During follow-up, a decrease in the cumulative frequency of all dominant clones correlated with a higher TIS.ConclusionIn treatment-naive patients with newly diagnosed IIM, muscle tissue and peripheral blood share expanded BcR clones. In our study a higher cumulative frequency of dominant BcR clones in blood before treatment was associated with a higher CK and better treatment response, suggesting that response to IVIG may depend on the composition of the pre-treatment BcR repertoire.

Contains fulltext : 229584.pdf (Publisher’s version ) (Open Access) Sjögren-Larsson syndrome (SLS) is a rare neurometabolic syndrome caused by deficient fatty aldehyde dehydrogenase. Patients exhibit intellectual disability, spastic paraplegia, and ichthyosis. The accumulation of fatty alcohols and fatty aldehydes has been demonstrated in plasma and skin but never in brain. Brain magnetic resonance imaging and spectroscopy studies, however, have shown an abundant lipid peak in the white matter of patients with SLS, suggesting lipid accumulation in the brain as well. Using histopathology, mass spectrometry imaging, and lipidomics, we studied the morphology and the lipidome of a postmortem brain of a 65-year-old female patient with genetically confirmed SLS and compared the results with a matched control brain. Histopathological analyses revealed structural white matter abnormalities with the presence of small lipid droplets, deficient myelin, and astrogliosis. Biochemically, severely disturbed lipid profiles were found in both white and gray matter of the SLS brain, with accumulation of fatty alcohols and ether lipids. Particularly, long-chain unsaturated ether lipid species accumulated, most prominently in white matter. Also, there was a striking accumulation of odd-chain fatty alcohols and odd-chain ether(phospho)lipids. Our results suggest that the central nervous system involvement in SLS is caused by the accumulation of fatty alcohols leading to a disbalance between ether lipid and glycero(phospho)lipid metabolism resulting in a profoundly disrupted brain lipidome. Our data show that SLS is not a pure leukoencephalopathy, but also a gray matter disease. Additionally, the histopathological abnormalities suggest that astrocytes and microglia might play a pivotal role in the underlying disease mechanism, possibly contributing to the impairment of myelin maintenance.

Circadian misalignment, as seen in shift work, is associated with an increased risk to develop type 2 diabetes. In an experimental setting, we recently showed that a rapid day-night shift for 3 consecutive nights leads to misalignment of the core molecular clock, induction of the PPAR pathway, and insulin resistance in skeletal muscle of young, healthy men. Here, we investigated if circadian misalignment affects the skeletal muscle lipidome and intramyocellular lipid droplet characteristics, explaining the misalignment-induced insulin resistance. Fourteen healthy men underwent one aligned and one circadian misalignment period, both consisting of similar to 3.5 days. In the misaligned condition, day and night were rapidly shifted by 12 hours leading to opposite eating, sleep, and activity times compared with the aligned condition. For each condition, two muscle biopsies were taken from the m. vastus lateralis in the morning and evening and subjected to semi-targeted lipidomics and confocal microscopy analysis. We found that only 2% of detected lipids were different between morning and evening in the aligned condition, whereas 12% displayed a morning-evening difference upon misalignment. Triacylglycerols, in particular species of a carbon length >= 55, were the most abundant lipid species changed upon misalignment. Cardiolipins were decreased upon misalignment, whereas phosphatidylcholines consistently followed the same morning-evening pattern, suggesting regulation by the circadian clock. Cholesteryl esters adjusted to the shifted behavior. Lipid droplet characteristics remained unaltered upon misalignment. Together, these findings show that simulated shift work disturbs the skeletal muscle lipidome, which may contribute to misalignment-induced insulin resistance.

Vaz, McDermott et al. identify variants in PCYT2, which encodes a key gene in phospholipid biosynthesis, in five individuals with a new complex hereditary spastic paraplegia. Functional studies in fibroblasts and a zebrafish model confirm the pathogenic nature of the variants, while lipidomic analysis reveals potential treatment strategies and plasma biomarkers., CTP:phosphoethanolamine cytidylyltransferase (ET), encoded by PCYT2, is the rate-limiting enzyme for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. Phosphatidylethanolamine is one of the most abundant membrane lipids and is particularly enriched in the brain. We identified five individuals with biallelic PCYT2 variants clinically characterized by global developmental delay with regression, spastic para- or tetraparesis, epilepsy and progressive cerebral and cerebellar atrophy. Using patient fibroblasts we demonstrated that these variants are hypomorphic, result in altered but residual ET protein levels and concomitant reduced enzyme activity without affecting mRNA levels. The significantly better survival of hypomorphic CRISPR-Cas9 generated pcyt2 zebrafish knockout compared to a complete knockout, in conjunction with previously described data on the Pcyt2 mouse model, indicates that complete loss of ET function may be incompatible with life in vertebrates. Lipidomic analysis revealed profound lipid abnormalities in patient fibroblasts impacting both neutral etherlipid and etherphospholipid metabolism. Plasma lipidomics studies also identified changes in etherlipids that have the potential to be used as biomarkers for ET deficiency. In conclusion, our data establish PCYT2 as a disease gene for a new complex hereditary spastic paraplegia and confirm that etherlipid homeostasis is important for the development and function of the brain.

Comprehensive metabolomic and lipidomic mass spectrometry methods are in increasing demand; for instance, in research related to nutrition and aging. The nematode Caenorhabditis elegans is a key model organism in these fields, owing to the large repository of available C. elegans mutants and their convenient natural lifespan. Here, we describe a robust and sensitive analytical method for the semi-quantitative analysis of >100 polar (metabolomics) and >1000 apolar (lipidomics) metabolites in C. elegans, using a single-sample preparation. Our method is capable of reliably detecting a wide variety of biologically relevant metabolic aberrations in, for example, glycolysis and the tricarboxylic acid cycle, pyrimidine metabolism and complex lipid biosynthesis. In conclusion, we provide a powerful analytical tool that maximizes metabolic data yield from a single sample. This article has an associated First Person interview with the joint first authors of the paper., Summary: We describe an integrated metabolomics and lipidomics method for analyzing metabolites in C. elegans using a single-sample preparation, which provides a robust way of obtaining comprehensive, high-quality metabolic data from biological samples.

ACBD5 deficiency is a novel peroxisome disorder with a largely uncharacterized pathology. ACBD5 was recently identified in a tethering complex mediating membrane contacts between peroxisomes and the endoplasmic reticulum (ER). An ACBD5-deficient mouse was analyzed to correlate ACBD5 tethering functions with the disease phenotype. ACBD5-deficient mice exhibit elevated very long-chain fatty acid levels and a progressive cerebellar pathology. Liver did not exhibit pathologic changes but increased peroxisome abundance and drastically reduced peroxisome-ER contacts. Lipidomics of liver and cerebellum revealed tissue-specific alterations in distinct lipid classes and subspecies. In line with the neurological pathology, unusual ultra-long chain fatty acids (C > 32) were elevated in phosphocholines from cerebelli but not liver indicating an organ-specific imbalance in fatty acid degradation and elongation pathways. By contrast, ether lipid formation was perturbed in liver towards an accumulation of alkyldiacylglycerols. The alterations in several lipid classes suggest that ACBD5, in addition to its acyl-CoA binding function, might maintain peroxisome-ER contacts in order to contribute to the regulation of anabolic and catabolic cellular lipid pathways., Darwisch, von Spangenberg et al. show that ACBD5‐deficient mice exhibit elevated levels of very long‐chain fatty acids and a progressive cerebellar pathology. A complex metabolic phenotype suggests that ACBD5 with its acyl‐CoA binding and peroxisome‐ER tethering functions might contribute to the regulation of anabolic and catabolic cellular lipid pathways.

Background Estrogen receptor (ER) positive breast cancer is often effectively treated with drugs that inhibit ER signaling, i.e., tamoxifen (TAM) and aromatase inhibitors (AIs). However, 30% of ER+ breast cancer patients develop resistance to therapy leading to tumour recurrence. Changes in the methylation profile have been implicated as one of the mechanisms through which therapy resistance develops. Therefore, we aimed to identify methylation loci associated with endocrine therapy resistance. Methods We used genome-wide DNA methylation profiles of primary ER+/HER2- tumours from The Cancer Genome Atlas in combination with curated data on survival and treatment to predict development of endocrine resistance. Association of individual DNA methylation markers with survival was assessed using Cox proportional hazards models in a cohort of ER+/HER2- tumours (N = 552) and two sub-cohorts corresponding to the endocrine treatment (AI or TAM) that patients received (N = 210 and N = 172, respectively). We also identified multivariable methylation signatures associated with survival using Cox proportional hazards models with elastic net regularization. Individual markers and multivariable signatures were compared with DNA methylation profiles generated in a time course experiment using the T47D ER+ breast cancer cell line treated with tamoxifen or deprived from estrogen. Results We identified 134, 5 and 1 CpGs for which DNA methylation is significantly associated with survival in the ER+/HER2-, TAM and AI cohorts respectively. Multi-locus signatures consisted of 203, 36 and 178 CpGs and showed a large overlap with the corresponding single-locus signatures. The methylation signatures were associated with survival independently of tumour stage, age, AI treatment, and luminal status. The single-locus signature for the TAM cohort was conserved among the loci that were differentially methylated in endocrine-resistant T47D cells. Similarly, multi-locus signatures for the ER+/HER2- and AI cohorts were conserved in endocrine-resistant T47D cells. Also at the gene set level, several sets related to endocrine therapy and resistance were enriched in both survival and T47D signatures. Conclusions We identified individual and multivariable DNA methylation markers associated with therapy resistance independently of luminal status. Our results suggest that these markers identified from primary tumours prior to endocrine treatment are associated with development of endocrine resistance.

Comprehensive metabolomic and lipidomic mass spectrometry methods are in increasing demand, for instance in research related to nutrition and aging. The nematode C. elegans is a key model organism in these fields, due to the large repository of available C. elegans mutants and their convenient natural lifespan. Here, we describe a robust and sensitive analytical method for the semi-quantitative analysis of >100 polar (metabolomics) and >1000 apolar (lipidomics) metabolites in C. elegans, using a single sample preparation. Our method is capable of reliably detecting a wide variety of biologically relevant metabolic aberrations in, for instance, glycolysis and the TCA cycle, pyrimidine metabolism and complex lipid biosynthesis. In conclusion, we provide a powerful analytical tool that maximizes metabolic data yield from a single sample.Graphical abstract

Breast cancer is one of the leading causes of death for women worldwide. Patients whose tumors express Estrogen Receptor α (ERα) account for around 70% of cases and are mostly treated with targeted endocrine therapy. However, 40% of these tumors eventually relapse due to resistance development and further treatment of these patients is highly ineffective. In this study we profiled the early phases of the resistance development process to uncover drivers of this phenomenon. Time-resolved analysis revealed that ATF3, a member of the ATF/CREB family of transcription factors, acts as a novel regulator of the response to therapy via rewiring of central signaling processes towards the adaptation to endocrine treatment. ATF3 was found to be essential in controlling crucial processes such as proliferation, cell cycle and apoptosis during the early response to treatment through the regulation of MAPK/AKT signaling pathways. Its essential role was confirmed in vivo in a mouse model and elevated expression of ATF3 was verified in patient datasets, adding clinical relevance to our findings. This study proposes ATF3 as a novel mediator of endocrine resistance development in breast cancer and elucidates its role in the regulation of downstream pathways activities.

Background The onset of seropositive rheumatoid arthritis (RA) is preceded by the presence of specific autoantibodies in the absence of synovial inflammation. Only a subset of these at-risk individuals will develop clinical disease. This impedes efforts to implement early interventions that may prevent onset of clinically manifest disease. Here we analyse whether clonal changes in the B cell receptor (BCR) repertoire can reliably predict onset of signs and symptoms. Methods In a prospective cohort study in 21 individuals at risk for RA based on the presence of autoantibodies, the BCR repertoire of paired peripheral blood and synovial tissue samples was analysed using next-generation BCR sequencing. BCR clones that were expanded beyond 0.5% of the total repertoire were labelled dominant. The relative risk (RR) for onset of arthritis was assessed using the presence of ≥5 dominant BCR clones as cut-off. Findings in peripheral blood were validated in an independent prospective cohort of 50 at-risk individuals. Based on the test cohort, individuals in the validation cohort were considered positive if peripheral blood at study entry showed ≥5 dominant BCR clones. Findings Both in the test and validation cohort, the presence of ≥5 dominant BCR clones in peripheral blood was significantly associated with arthritis development after follow-up (validation cohort RR 6.3, 95% CI 2.7 to 15, p −4 ). Even when adjusted for a recently described clinical prediction rule the association remained intact (RR 5.0, 95% CI 1.2 to 20, p=0.024). When individuals developed arthritis, dominant BCR clones disappeared from peripheral blood and appeared in synovial tissue, suggesting a direct role of these clones in disease pathogenesis. Interpretation Dominant BCR clones in peripheral blood predict onset of clinical signs and symptoms of RA in at-risk individuals with high accuracy. Our data suggest that during onset of RA these clones shift from peripheral blood to the target tissue.

Birth weight (BW) variation is influenced by fetal and maternal genetic and non-genetic factors, and has been reproducibly associated with future cardio-metabolic health outcomes. These associations have been proposed to reflect the lifelong consequences of an adverse intrauterine environment. In earlier work, we demonstrated that much of the negative correlation between BW and adult cardio-metabolic traits could instead be attributable to shared genetic effects. However, that work and other previous studies did not systematically distinguish the direct effects of an individual’s own genotype on BW and subsequent disease risk from indirect effects of their mother’s correlated genotype, mediated by the intrauterine environment. Here, we describe expanded genome-wide association analyses of own BW (n=321,223) and offspring BW (n=230,069 mothers), which identified 278 independent association signals influencing BW (214 novel). We used structural equation modelling to decompose the contributions of direct fetal and indirect maternal genetic influences on BW, implicating fetal- and maternal-specific mechanisms. We used Mendelian randomization to explore the causal relationships between factors influencing BW through fetal or maternal routes, for example, glycemic traits and blood pressure. Direct fetal genotype effects dominate the shared genetic contribution to the association between lower BW and higher type 2 diabetes risk, whereas the relationship between lower BW and higher later blood pressure (BP) is driven by a combination of indirect maternal and direct fetal genetic effects: indirect effects of maternal BP-raising genotypes act to reduce offspring BW, but only direct fetal genotype effects (once inherited) increase the offspring’s later BP. Instrumental variable analysis using maternal BW-lowering genotypes to proxy for an adverse intrauterine environment provided no evidence that it causally raises offspring BP. In successfully separating fetal from maternal genetic effects, this work represents an important advance in genetic studies of perinatal outcomes, and shows that the association between lower BW and higher adult BP is attributable to genetic effects, and not to intrauterine programming.

Objective To gain more insight into the dynamics of lymphocyte depletion and develop new predictors of clinical response to rituximab in rheumatoid arthritis (RA). Methods RNA-based next-generation sequencing was used to analyse the B cell receptor (BCR) repertoire in peripheral blood and synovial tissue samples collected from 24 seropositive patients with RA treated with rituximab. Clonal expansion, mutation load and clonal overlap were assessed in samples collected before, at week 4 and at week 16 or 24 after treatment and correlated to the patients’ clinical response. Results After 4 weeks of rituximab-induced B cell depletion, the peripheral blood BCR repertoire of treated patients consisted of fewer, more dominant and more mutated BCR clones. No significant changes in the synovial tissue BCR repertoire were detected until week 16 post-treatment, when a reduced clonal overlap with baseline and an increased mutation load were observed. In patients who were non-responders at month 3 (n=5) using the European League Against Rheumatism response criteria, peripheral blood samples taken at week 4 after rituximab treatment showed more dominant clones compared with moderate responders (n=9) (median (IQR): 36 (27–52) vs 18 (16–26); p Conclusion Significant changes in BCR clonality are observed in peripheral blood of patients 4 weeks after rituximab treatment, while changes in synovial tissue were observed at later time points. Incomplete depletion of the dominant baseline peripheral blood BCR repertoire in the first month of treatment might predict clinical non-response at 3 months.

Cell Metabolism, 31 (3), ISSN:1550-4131, ISSN:1932-7420

Genetic and immunological evidence clearly points to a role for T cells in the pathogenesis of rheumatoid arthritis (RA). Selective targeting of such disease-associated T cell clones might be highly effective while having few side effects. However, such selective targeting may only be feasible if the same T cell clones dominate the immune response at different sites of inflammation. We leveraged high-throughput technology to quantitatively assess whether different T cell clones dominate the inflammatory infiltrate at various sites of inflammation in this prototypic autoimmune disease. In 13 RA patients, we performed quantitative next-generation sequencing–based human TCRβ repertoire analysis in simultaneously obtained samples from inflamed synovial tissue (ST) from distinct locations within one joint, from multiple joints, and from synovial fluid (SF) and peripheral blood (PB). Identical TCRβ clones dominate inflammatory responses in ST samples taken from different locations within a single joint and when sampled in different joints. Although overall ST–SF overlap was comparable to higher ST–ST values, the overlap in dominant TCRβ clones in ST–SF comparisons was much lower than ST–ST and comparable to the low ST–PB overlap. In individual RA patients, a limited number of TCRβ clones dominate the immune response in the inflamed ST regardless of the location within a joint and which joint undergoes biopsy; in contrast, there is limited overlap of ST with SF or PB TCR repertoires. This limited breadth of the T cell response in ST of the individual RA patient indicates that development of immunotherapies that selectively modulate dominant T cell responses might be feasible.

Peroxisomes are ubiquitous cell organelles that play an important role in lipid metabolism. Accordingly, peroxisomal disorders, including the peroxisome biogenesis disorders and peroxisomal single-enzyme deficiencies, are associated with aberrant lipid metabolism. Lipidomics is an emerging tool for diagnosis, disease-monitoring, identifying lipid biomarkers, and studying the underlying pathophysiology in disorders of lipid metabolism. In this study, we demonstrate the potential of lipidomics for the diagnosis of peroxisomal disorders using plasma samples from patients with different types of peroxisomal disorders. We show that the changes in the plasma profiles of phospholipids, di- and triglycerides, and cholesterol esters correspond with the characteristic metabolite abnormalities that are currently used in the metabolic screening for peroxisomal disorders. The lipidomics approach, however, gives a much more detailed overview of the metabolic changes that occur in the lipidome. Furthermore, we identified novel unique lipid species for specific peroxisomal diseases that are candidate biomarkers. The results presented in this paper show the power of lipidomics approaches to enable the specific diagnosis of different peroxisomal disorders. Electronic supplementary material The online version of this article (10.1007/s10545-017-0114-7) contains supplementary material, which is available to authorized users.

Peroxisomes play an important role in a variety of metabolic pathways, including the α- and β-oxidation of fatty acids, and the biosynthesis of ether phospholipids. Single peroxisomal enzyme deficiencies (PEDs) are a group of peroxisomal disorders in which either a peroxisomal matrix enzyme or a peroxisomal membrane transporter protein is deficient. To investigate the functional consequences of specific enzyme deficiencies on the lipidome, we performed lipidomics using cultured skin fibroblasts with different defects in the β-oxidation of very long-chain fatty acids, including ABCD1- (ALD), acyl-CoA oxidase 1 (ACOX1)-, D-bifunctional protein (DBP)-, and acyl-CoA binding domain containing protein 5 (ACBD5)-deficient cell lines. Ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry revealed characteristic changes in the phospholipid composition in fibroblasts with different fatty acid β-oxidation defects. Remarkably, we found that ether phospholipids, including plasmalogens, were decreased. We defined specific phospholipid ratios reflecting the different enzyme defects, which can be used to discriminate the PED fibroblasts from healthy control cells. Electronic supplementary material The online version of this article (doi:10.1007/s10545-017-0076-9) contains supplementary material, which is available to authorized users.

Short hairpin RNAs (shRNAs) are widely used for gene knockdown by inducing the RNA interference (RNAi) mechanism, both for research and therapeutic purposes. The shRNA precursor is processed by the RNase III-like enzyme Dicer into biologically active small interfering RNA (siRNA). This effector molecule subsequently targets a complementary mRNA for destruction via the Argonaute 2 (AGO2) complex. The cellular role of Dicer concerns the processing of pre-miRNAs into mature microRNA (miRNA). Recently, a non-canonical pathway was reported for the biogenesis of miR-451, which bypasses Dicer and is processed instead by the slicer activity of AGO2, followed by the regular AGO2-mediated mRNA targeting step. Interestingly, shRNA designs that are characterized by a relatively short basepaired stem also bypass Dicer to be processed by AGO2. We named this design AgoshRNA as these molecules depend on AGO2 both for processing and silencing activity. In this study, we investigated diverse mechanistic aspects of this new class of AgoshRNA molecules. We probed the requirements for AGO2-mediated processing of AgoshRNAs by modification of the proposed cleavage site in the hairpin. We demonstrate by deep sequencing that AGO2-processed AgoshRNAs produce RNA effector molecules with more discrete ends than the products of the regular shRNA design. Furthermore, we tested whether trimming and tailing occurs upon AGO2-mediated processing of AgoshRNAs, similar to what has been described for miR-451. Finally, we tested the prediction that AgoshRNA activity, unlike that of regular shRNAs, is maintained in Dicer-deficient cell types. These mechanistic insights could aid in the design of optimised AgoshRNA tools and therapeutics.

The technical advances in mass spectrometry, particularly the development of (ultra)-high-resolution/mass accuracy measurement capabilities in combination with refinement of soft ionization techniques, have increased the application and success of lipidomics to answer biological questions in relation to lipid metabolism. Together with other omics technologies, lipidomics has become an important tool to practice systems biology as lipids comprise a very significant part of the metabolome and play pleiotropic roles in cellular functions. As an increasing number of disorders are linked to lipid metabolism, lipidomics is used to search for biomarkers, understand disease mechanism and follow the efficacy of therapeutic options. This review provides a first introduction to the major methodological strategies currently used for mass spectrometry-based lipidomics and associated data pre-processing and analysis.

Human cytomegalovirus (hCMV) infection is characterized by a vast expansion of resting effector-type virus-specific T cells in the circulation. In mice, interleukin-7 receptor α (IL-7Rα)-expressing cells contain the precursors for long-lived antigen-experienced CD8 + T cells, but it is unclear if similar mechanisms operate to maintain these pools in humans. Here, we studied whether IL-7Rα-expressing cells obtained from peripheral blood (PB) or lymph nodes (LNs) sustain the circulating effector-type hCMV-specific pool. Using flow cytometry and functional assays, we found that the IL-7Rα + hCMV-specific T cell population comprises cells that have a memory phenotype and lack effector features. We used next-generation sequencing of the T cell receptor to compare the clonal repertoires of IL-7Rα + and IL-7Rα − subsets. We observed limited overlap of clones between these subsets during acute infection and after 1 year. When we compared the hCMV-specific repertoire between PB and paired LNs, we found many identical clones but also clones that were exclusively found in either compartment. New clones that were found in PB during antigenic recall were only rarely identical to the unique LN clones. Thus, although PB IL-7Rα-expressing and LN hCMV-specific CD8 + T cells show typical traits of memory-type cells, these populations do not seem to contain the precursors for the novel hCMV-specific CD8 + T cell pool during latency or upon antigen recall. IL-7Rα + PB and LN hCMV-specific memory cells form separate virus-specific compartments, and precursors for these novel PB hCMV-specific CD8 + effector-type T cells are possibly located in other secondary lymphoid tissues or are being recruited from the naive CD8 + T cell pool. IMPORTANCE Insight into the self-renewal properties of long-lived memory CD8 + T cells and their location is crucial for the development of both passive and active vaccination strategies. Human CMV infection is characterized by a vast expansion of resting effector-type cells. It is, however, not known how this population is maintained. We here investigated two possible compartments for effector-type cell precursors: circulating acute-phase IL-7Rα-expressing hCMV-specific CD8 + T cells and lymph node (LN)-residing hCMV-specific (central) memory cells. We show that new clones that appear after primary hCMV infection or during hCMV reactivation seldom originate from either compartment. Thus, although identical clones may be maintained by either memory population, the precursors of the novel clones are probably located in other (secondary) lymphoid tissues or are recruited from the naive CD8 + T cell pool.

Understanding cellular adaptation to environmental changes is one of the major challenges in systems biology. To understand how cellular systems react towards perturbations of their steady state, the metabolic dynamics have to be described. Dynamic properties can be studied with kinetic models but development of such models is hampered by limited in vivo information, especially kinetic parameters. Therefore, there is a need for mathematical frameworks that use a minimal amount of kinetic information. One of these frameworks is dynamic flux balance analysis (DFBA), a method based on the assumption that cellular metabolism has evolved towards optimal changes to perturbations. However, DFBA has some limitations. It is less suitable for larger systems because of the high number of parameters to estimate and the computational complexity. In this paper, we propose MetDFBA, a modification of DFBA, that incorporates measured time series of both intracellular and extracellular metabolite concentrations, in order to reduce both the number of parameters to estimate and the computational complexity. MetDFBA can be used to estimate dynamic flux profiles and, in addition, test hypotheses about metabolic regulation. In a first case study, we demonstrate the validity of our method by comparing our results to flux estimations based on dynamic 13C MFA measurements, which we considered as experimental reference. For these estimations time-resolved metabolomics data from a feast-famine experiment with Penicillium chrysogenum was used. In a second case study, we used time-resolved metabolomics data from glucose pulse experiments during aerobic growth of Saccharomyces cerevisiae to test various metabolic objectives.

Use of some HIV-1 nucleoside reverse transcriptase inhibitors (NRTI) is associated with severe adverse events. However, the exact mechanisms behind their toxicity has not been fully understood. Mitochondrial dysfunction after chronic exposure to specific NRTIs has predominantly been assigned to mitochondrial polymerase-γ inhibition by NRTIs. However, an increasing amount of data suggests that this is not the sole mechanism. Many NRTI induced adverse events have been linked to the incurrence of oxidative stress, although the causality of events leading to reactive oxygen species (ROS) production and their role in toxicity is unclear. In this study we show that short-term effects of first generation NRTIs, which are rarely discussed in the literature, include inhibition of oxygen consumption, decreased ATP levels and increased ROS production. Collectively these events affect fitness and longevity of C. elegans through mitohormetic signalling events. Furthermore, we demonstrate that these effects can be normalized by addition of the anti-oxidant N-acetylcysteine (NAC), which suggests that ROS likely influence the onset and severity of adverse events upon drug exposure.